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Exploring Yeast As a Cell Factory for the Production of Carboxylic Acids and Derivatives Exploring Yeast as a Cell Factory for the Production of Carboxylic Acids and Derivatives Portugal-Nunes, Diogo 2017 Document Version: Publisher's PDF, also known as Version of record Link to publication Citation for published version (APA): Portugal-Nunes, D. (2017). Exploring Yeast as a Cell Factory for the Production of Carboxylic Acids and Derivatives. Department of Chemistry, Lund University. Total number of authors: 1 Creative Commons License: Unspecified General rights Unless other specific re-use rights are stated the following general rights apply: Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Read more about Creative commons licenses: https://creativecommons.org/licenses/ Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. LUND UNIVERSITY PO Box 117 221 00 Lund +46 46-222 00 00 Exploring Yeast as a Cell Factory for the Production of Carboxylic Acids and Derivatives APPLIED MICROBIOLOGY | FACULTY OF ENGINEERING | LUND UNIVERSITY DIOGO PORTUGAL-NUNES Exploring Yeast as a Cell Factory for the Production of Carboxylic Acids and Derivatives Diogo Portugal-Nunes DOCTORAL DISSERTATION which, by due permission of the Faculty of Engineering, Lund University, Sweden, will be publicly defended on 16th June 2017 at 10:00 in Lecture Hall B, Kemicentrum, Naturvetarvägen 12-18, Lund, for the degree of Doctor of Philosophy. Faculty opponent Prof. Stéphane Guillouet Fermentation Advances and Microbial Engineering, Department of Biochemical Engineering, Institut National des Sciences Appliquées de Toulouse, France i Organization Document name DOCTORAL DISSERTATION LUND UNIVERSITY Division of Applied Microbiology Date of issue: June 16th, 2017 Author: Diogo João Portugal-Nunes Sponsoring organization: EU projects BRIGIT (no. 311935) and BioREFINE-2G (no. 613771) Title: Exploring Yeast as a Cell Factory for the Production of Carboxylic Acids and Derivatives Abstract Baker’s yeast, Saccharomyces cerevisiae, is a promising cell factory for the sustainable utilization of renewable resources for the formation of products with commercial value. Among these, poly-3-D-hydroxybutyrate (PHB) is an extensively studied biopolymer naturally accumulated in some bacteria and archaea species through the formation of carbon granules. Its bio-based origin, biodegradability and applications in several industries makes it one of the most interesting biopolymers. In the present study, aerobic production of PHB from xylose was achieved in S. cerevisiae through the engineering of an optimized xylose oxido-reducing pathway and the expression of the genes involved in the PHB-producing pathway from the bacterium Cupriavidus necator. As anaerobicity is generally preferred in industrial applications, leading to an excess of NADH in the yeast metabolism, S. cerevisiae was further engineered by the introduction of a NADH-dependent acetoacetyl-CoA reductase from the bacterium Allochromatium vinosum. PHB formation clearly benefited from this modification and its formation from pure carbon sources under both anaerobic and oxygen-limited conditions was observed. The influence of nitrogen availability on PHB accumulation was also investigated. In contrast to the natural producers, PHB formation in S. cerevisiae was favored by high levels of nitrogen. These engineering strategies together resulted in one of the highest PHB contents reported in S. cerevisiae to date. The production of carboxylic acids, i.e. organic compounds that can be used as building blocks for a wide range of products, was also investigated in yeast due to its robustness and ability to grow at low pH. Cytosolic production of alpha-ketoglutarate (AKG) from xylose was attempted by rewiring the carbon flux towards the glyoxylate cycle in S. cerevisiae. Although AKG production was low, the study contributed to a deeper understanding of the mitochondrial and cytosolic formation of carboxylic acids in S. cerevisiae, revealing novel routes for their bio-production and for further optimization studies. In the last part of this work, AKG production was attempted by using a heterologous oxidative pathway that bypasses glycolysis and links xylose directly to the tricarboxylic acid cycle – the so-called Weimberg pathway. The Weimberg pathway was found to be partially active and highlighted the fact that the assembly and activity of the proteins converting xylonate into AKG require further development. Keywords: Saccharomyces cerevisiae, poly-3-D-hydroxybutyrate (PHB), carboxylic acids, alpha-ketoglutarate (AKG), xylose assimilation, Weimberg pathway Classification system and/or index terms (if any) Supplementary bibliographical information Language: English ISSN and key title ISBN 978-91-7422-524-2 (printed version) 978-91-7422-525-9 (digital version) Recipient’s notes Number of pages: 196 Price Security classification I, the undersigned, being the copyright owner of the abstract of the above-mentioned dissertation, hereby grant to all reference sources permission to publish and disseminate the abstract of the above-mentioned dissertation. Signature: Date: April 26th, 2017 ii Exploring Yeast as a Cell Factory for the Production of Carboxylic Acids and Derivatives Diogo Portugal-Nunes iii FrontFront cover: cover: Illustration Illustration by by Diogo Diogo Portugal-Nunes Portugal-Nunes BackBack cover: cover: Drawing Drawing by by Diogo Diogo Portugal-Nunes Portugal-Nunes and and photo photo taken taken by by Yasmine Yasmine Akel Akel ©© Diogo Diogo Portugal-Nunes Portugal-Nunes DivisionDivision of of Applied Applied Microbiology Microbiology DepartmentDepartment of of Chemistry Chemistry FacultyFaculty of of Engineering Engineering P.O.P.O. Box Box 124 124 SE-221SE-221 00 00 Lund Lund SwedenSweden ISBN:ISBN: 978-91-7422-524-2 978-91-7422-524-2 (printed (printed version) version) ISBN:ISBN: 978-91-7422-525-9 978-91-7422-525-9 (digital (digital version) version) PrintedPrinted in in Sweden Sweden by by Media-Tryck, Media-Tryck, Lund Lund University University LundLund 2017 2017 Media-Tryck is an environmentally certified and ISO 14001 certified provider of printed material. Read more about our environmental work at www.mediatryck.lu.se iviv Para os meus pais v vi Abstract Baker’s yeast, Saccharomyces cerevisiae, is a promising cell factory for the sustainable utilization of renewable resources for the formation of products with commercial value. Among these, poly-3-D-hydroxybutyrate (PHB) is an extensively studied biopolymer naturally accumulated in some bacteria and archaea species through the formation of carbon granules. Its bio-based origin, biodegradability and applications in several industries makes it one of the most interesting biopolymers. In the present study, aerobic production of PHB from xylose was achieved in S. cerevisiae through the engineering of an optimized xylose oxido-reducing pathway and the expression of the genes involved in the PHB-producing pathway from the bacterium Cupriavidus necator. As anaerobicity is generally preferred in industrial applications, leading to an excess of NADH in the yeast metabolism, S. cerevisiae was further engineered by the introduction of an NADH-dependent acetoacetyl-CoA reductase from the bacterium Allochromatium vinosum. PHB formation clearly benefited from this modification and its formation from pure carbon sources under both anaerobic and oxygen-limited conditions was observed. The influence of nitrogen availability on PHB accumulation was also investigated. In contrast to the natural producers, PHB formation in S. cerevisiae was favored by high levels of nitrogen. These engineering strategies together resulted in one of the highest PHB contents reported in S. cerevisiae to date. The production of carboxylic acids, i.e. organic compounds that can be used as building blocks for a wide range of products, was also investigated in yeast due to its robustness and ability to grow at low pH. Cytosolic production of alpha-ketoglutarate (AKG) from xylose was attempted by rewiring the carbon flux towards the glyoxylate cycle in S. cerevisiae. Although AKG production was low, the study contributed to a deeper understanding of the mitochondrial and cytosolic formation of carboxylic acids in S. cerevisiae, revealing novel routes for their bio-production and for further optimization studies. In the last part of this work, AKG production was attempted by using a heterologous oxidative pathway that bypasses glycolysis and links xylose directly to the tricarboxylic acid cycle – the so-called Weimberg pathway. The Weimberg pathway was found to be partially active and highlighted the fact that the assembly and activity of the proteins converting xylonate into AKG require further development. vii viii Popular scientific summary Microorganisms, or microbes, are microscopic living organisms with great diversity. It has recently been estimated that 300 trillion different microbial species are present
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